Polysialic acid (PSA), known as colominic acid, is a polymer of sialic acid whose degree of polymerization (DP) is 8 to 200 residues (Barker, S. A., Jones, R. G., Somers, P. J.; Improvements in the production and isolation of Colominic acid; Carbohydrate research; 369-376, 3, (1967). The most common structure of PSA is the Neu5Ac polymer whose inter-residual linkage is ∝2→8. The conformation of PSA is a helical structure in which N-acetyl substitution of the C5 position faces the outer space of the helix. PSA occur as important components of glycoprotein, gangliosides and oligosaccharides and are usually found in their terminal positions.
PSA is a natural constituent of the human body and also of certain bacteria. PSA evolved over a millions of years by bacteria to foil the body's defense system. Being chemically identical to PSA in the human body, bacterial PSA, by virtue of this structural mimicry, is completely non-immunogenic, even when coupled to proteins. Unlike other polymers (e.g. PEG) PSA is biodegradable. This is particularly important where a polymer is used for therapeutics given chronically or in large dosages.
Polysialic acid has extensive applications in the pharmacology. Exclusive presence of therapeutics in a stable form in the body, is often needed for optimal use. However, many therapeutics, for instance cytokines, hormones, enzymes and other proteins such as antibody fragments, as well as conventional drugs, are inactivated or removed from the body rapidly and before effective concentrations in the blood or target tissues can be attained. In this respect, polysialic acid can be used to improve the pharmacokinetics and pharmacodynamics of therapeutic molecules. PSA as a delivery vehicle for drug molecules particularly of small sizes can circumvent many of the problems encountered in the direct use of such drug molecules. It can preserve functionality on conjugation, improve stability in vivo, prolong presence in the blood circulation, prolong pharmacological action, reduce immunogenicity and antigenicity. PSA has certain advantages that may be essential for the next generation of therapeutics. Particularly high molecular weight PSA will be desirable to act as vehicle for drug molecules.
In order to meet the increasing demand for commercial PSA, economical large-scale production of high-grade purity PSA is necessary. Only a few natural sources such as swallow's nests, whey from cheese production, chalaze, egg membranes and the products of microbial fermentation processes, have been explored for practical industrial scale preparation of PSA.
JP 1-144989A discloses the production of colominic acid by E. coli M12 but the yields were very low ˜40 mg/L.
JP 05084091 discloses the use of L-malic acid as a source of C and the ammonium sulfate as a source of N, in order to increase the production of the colominic acid. The patent does not disclose the final yields, process of isolation and purity of the product.
JP 06245786A2 discloses fermentation of liquid raw material using E. coli in order to yield the colominic acid and further purified by affinity column chromatography by using lectin as a stationary phase. The drawback associated with such method is that it involves multi-step lengthy process of immobilizing lectin over gel, for use as a stationary phase. Also, during further purification process it requires additional elution steps with sodium acetate buffer to clear N-acetyl-D-glucosamine used as an inhibition agent for wheat germ lectin and glycoprotein.
Mushtaq et. al. (2004 Antimicrob Agents Chemother. 48(5) 1503-1508) disclosed E. coli K1 LP 1674 producing PSA wherein the E. coli produces the PSA in body of the patient to cause infection.
Weisgerber et. al. (1990 JBC 265/3 1578-1587) and Pelkonen et. al. (1988 J. Bacteriol. 170 (6) 2646-2653) disclosed media containing casamino acid for the production of PSA. The article discussed biosynthesis of PSA capsule in E. coli K1. All the work done here is on laboratory/characterization scale.
Zhan et. al. (2002 Biochem. Engg J. 11(2) 201-204) disclosed production of PSA by batch and fed-batch fermentation with controlled pH 6.4 during stationary phase. The drawback of this method is that the PSA produced by this method is of lower molecular weight.
Ringenberg et. al. (2001 Glycobiology 11(7) 533-539) disclosed isolation of PSA using Ultrafiltration and analysis using HPLC on CarboPac PA 1 column. The volumes handles here were in mu. L level.
Lin et. al. (1999 Glycobiology 9(8) 807-814) and Inoue et. al. (2001 Glycobiology 11(9) 759-769) disclosed preparation of samples of oligo/polysialic acid using Prep DEAE Sephadex A 25 wherein pure samples of colominic acid or Ne5Ac were obtained from various sources which were then separated using preparative ion exchange chromatography.
Ohe et. al. (2002 Glycobiology 12(1) 47-63), Miyata et. al. (2004 Glycobiology 14(9) 827-840) and Hallenbeck et. al. (1987 Anal Biochem. Feb 15; 161(1):181-6) disclosed HPLC analysis of PSA samples using anion exchange column.
JP08-070882 by NGK Insulators Ltd disclosed process for purification of sialic acid after hydrolysis of PSA, wherein the PSA was isolated using ultrafiltration and ion exchange chromatography. The patent does not disclose yields of PSA and the purity of the same. The scale of the operation is also not clear.
Adam et. al. 1995 Anal Biochem. Mar 1; 225(2):321-7 disclosed that ultracentrifugation, Detoxi-Gel, and ion-exchange chromatography did not remove endotoxin, except gel filtration chromatography performed at 60 degrees C. in sodium deoxycholate buffer.
For removal of DNA, protein, lipid and other impurities, purification processes like phenol extraction, detergents and solvent precipitation etc. are used. The disadvantages of phenol is that it is corrosive and unsafe to handle during scale up; The extraction and phase separation procedure with solvents are tedious using scaled up equipments with GMP, handling large quantity of phenol at 65° C. is inconvenient in extraction procedure.
Other purification method involving precipitation has the drawbacks of inconvenient and tedious operation at commercial scale, batch-to-batch inconsistency, low recovery and low degree of purification.
Trichloroacetic acid (TCA) is commonly used to precipitate the proteins; however TCA can hydrolyze the products like polysialic acid during the purification steps.
Use of CTAB detergent for precipitation [(Bolanos, R., Dewitt, C. W., Isolation and characterization of the K1 (L) antigen of Escherichia coli, J. Bacteriology. 1987-1996, 19, 3, (1966)] has drawback of poor yields. It is also difficult to remove detergents which interferes with other purification method hence require an additional step of removal. Also an additional step in the form of ethanol precipitation further lowers the recovery due to simultaneous precipitation of product like PSA and protein Additional step of re-dissolving the precipitate and fractional precipitation steps have to be added.
Phenyl boronate is used, to remove carbohydrate impurities in the chromatography process, which is known to have general affinity for carbohydrates (Hage, D. S., Affinity chromatography: A review of clinical applications, Clin. Chem. 593-615, 45:5, (1999). The column requires higher quantities of resin during scale up. The major difficulty in using Phenyl boronate is that it retains the protein impurities in a mixture without separation and thus affecting the purity.
It is reported in literature that hydroxylapetitie (HA) is used for the separation of DNA from samples as it shows high binding affinity for DNA molecules. The disadvantage proteins also remain along with DNA beyond the permissible limits.
Few possible processing steps would result in having the most efficient way of reaching high process efficiency and low costs in the overall production process. Most currently used purification processes still involve multiple steps of processing which add to the costs, loss of product and offer opportunities for contamination.
The prior art disclosed above suffers from one or more drawbacks selected from:
1. Analytical or preparative scale methods
2. Lower yields
3. Crude or impure product
Therefore, a need exists for a process for production and purification of PSA that overcomes the problems seen in the prior art and provides higher yields of Polysialic acid with high-grade purity.